mRNA vaccines have attracted significant interest recently due to their success during the COVID-19 pandemic. Their success is attributed to advances in the design and encapsulation of mRNA into ionizable lipid nanoparticles (iLNPs). These vaccines are based on mRNA encapsulated in iLNPs, which are approximately 100 nm in diameter, and have a neutral surface charge and a dense electron-rich oily core. iLNP is currently the most clinically advanced mRNA delivery vehicle. In order to apply mRNA to these other areas, further optimization and development of iLNPs and mRNA may be required. Therefore, a method that can be used to make mRNA-iLNPs could allow more researchers to evaluate and advance them.
Recently, McKenzie RE et al at the Victoria University of Wellington in New Zealand describe the production of mRNA-iLNPs, from the synthesis of mRNA to the formulation and characterization of the iLNP.
- Basic Protocol 1 describes a 3-step method for the synthesis of mRNA from a DNA template by in vitro transcription (IVT), followed by enzymatic capping and tailing. This method allows independent study of each component of the mRNA molecule [coding region, non-coding cis-regulatory region, 5' cap and 3' poly(A) tail] and is the most efficient capping method (100% capped mRNA after enzymatic treatment) and avoids the difficulty of poly(A) tract instability during DNA template production.
- Basic Protocol 2 describes the formulation of mRNA in iLNPs using a microfluidic mixer. The alternative protocol outlines the process of formulating small amounts of mRNA by complexing the mRNA into preformed vesicles and downstream processing.
- Basic Protocol 3 covers the characterization of iLNPs, including routine analysis of size, polydispersity, charge, and encapsulation efficiency, as well as less routine analysis of apparent particle pKa and mRNA quality after encapsulation. The protocol can be adapted to different lipids, payloads, and formulation parameters as needed.
Figure 1. Summary of protocols.
DNA Template
The DNA template should be linear and can be generated by linearization of purified plasmids or by PCR. This template requires the T7 promoter sequence 5' TAATACGACTCACTATA 3' to allow T7 RNA polymerase to initiate in vitro transcription (IVT), and should encode the antigen of interest in an open reading frame (ORF) flanked by a 5′ and 3′ untranslated region (UTR), if desired. Before mRNA production, it is necessary to calculate how much DNA template is required for IVT. For example, in Basic Protocol 1, preparing 150 μg of mRNA requires ~1 μg of linearized template, and this amount should be increased accordingly.
Modified Nucleoside Triphosphates
Modified nucleoside triphosphates (NTPs) such as pseudouridine (Ѱ), N1-methyl-pseudouridine (m1Ѱ) and 5-methoxyuridine (5moU) reduce the innate immune stimulating properties of mRNA because they have less specificity to RNA-sensing receptors. This reduces type I IFN production, improves transfection, translation efficiency, and reduces toxicity. Complete or partial replacement can be performed, with immunosuppression increasing proportionally with the proportion of modified NTP. For vaccines, the modified/unmodified NTP ratio needs to be optimized by the user for the specific application, route of administration, and iLNP formulation.
mRNA Storage
Unnecessary freezing and thawing of mRNA should be avoided. The final product should be frozen in small single-serve aliquots and thawed only when needed. The mRNA can be stored long-term in water at –80°C or in a low pH solution suitable for encapsulation (such as sodium acetate).
Encapsulating mRNA in Ionizable Lipid Nanoparticles
Encapsulation of mRNA should occur within 1 day. iLNPs are best used fresh, so formulations should be planned close to their use-by date.
Scale
When planning the scale of mRNA synthesis, the amount of encapsulated mRNA required should be considered. Using the Basic Protocol 2 requires approximately 300 µg of mRNA to return 100 µg of encapsulated mRNA, while using the Alternative Protocol requires 150 µg to achieve the same yield.
mRNA Purification Methods
After each of the three steps of mRNA synthesis (IVT, capping, and tailing), a purification step is required to isolate the mRNA product of interest and remove reaction components such as buffers, enzymes, and NTPs. For less experienced users, RNA cleanup kits are available. These alternative clean-up steps may also be preferred when time is limited and overnight precipitation after the capping and tailing steps are not convenient.
Reference
McKenzie RE, Minnell JJ, Ganley M, Painter GF, Draper SL. mRNA Synthesis and Encapsulation in Ionizable Lipid Nanoparticles. Curr Protoc. 2023 Sep;3(9):e898.